Historically, the detection and identification of bacteria, mycobacteria, yeasts, and molds have relied primarily on their morphologic and phenotypic properties. This approach is imprecise and slow for many clinically significant microbes. For this reason, we have explored alternative methods, such as Sanger sequencing, pyrosequencing, and mass spectroscopy, for the detection and identification of selected organisms. Earlier studies from our lab demonstrated that Sanger sequencing and pyrosequencing were powerful tools for the definitive identification of bacteria including Nocardia and Mycobacterium, yeasts, and molds; however, the procedures are expensive and time-consuming. Studies in the current fiscal year have focused on alternative identification methods, specifically MALDI-TOF (matrix-assisted laser desorption-ionization time of flight mass spectrometry) and most recently Raman spectroscopy. MALDI-TOF MS provides a reproducible spectral pattern based on the mass/charge (m/z) ratio of ionized proteins. Preparation of samples is rapid and inexpensive. Colony material is washed with ethanol, extracted with 1:1 Formic Acid (70%):Acetonitrile, and spotted on MALDI plates with cyano-4-hydroxycinnamic acid as the matrix. Up to 396 samples can be evaluated in a single run, with the identification of individual organisms requiring approximately 5 minutes. A series of experiments have been performed with well-characterized reference strains of yeasts, Nocardia, and bacteria used to build a clinical database and then challenged with clinical isolates. For yeasts, 105 type and reference strains were used to build the database, and then challenged with 115 unique clinical isolates. A total of 95% of the clinical isolates were correctly identified, with no identification obtained for 5% of the isolates. For Nocardia 54 type and reference strains were used to build the database for 47 Nocardia species, and then 80 clinical strains were used to challenge the database. A total of 74% of the strains were identified at the species level with the remainder identified at the group level. No Nocardia isolates were misidentified. Finally, studies were performed to determine if MALDI-OF MS could be used to identify bacteria and yeasts observed in positive blood culture broths. A method for concentrating bacteria in positive blood culture broths and eliminating blood cells and serum proteins was developed. A total of 210 positive cultures were examined representing 32 genera and 59 species/groups. Using a spectral score of >1.7, 75.8% of the blood cultures isolates were identified correctly at the species level, no identification (spectral score <1.7) was obtained for 20% of the isolates, one organism (Salmonella) was identified at the genus level, and 8 isolates of Streptococcus mitis were misidentified as Streptococcus pneumoniae. The lack of identification for 20% of the isolates was due to insufficient organisms present in the sample, and the misidentification of S. mitis as the closely related species, S. pneumoniae, was recognized as a limitation of this application. Work is continuing on refining bacterial identification and extending this research into identification schemes for mycobacteria and molds. Three publications are currently under review related to these studies. Preliminary studies have just been initiated using Raman spectrometry for typing bacterial isolates, such as Acinetobacter baumannii, vancomycin-resistant enterococci, and oxacillin-resistant Staphylococcus aureus.